Summary
The Gram‐positive bacterium Bacillus subtilis uses serine not only as a building block for proteins but also as an important precursor in many anabolic reactions. Moreover, a lack of serine ...results in the initiation of biofilm formation. However, excess serine inhibits the growth of B. subtilis. To unravel the underlying mechanisms, we isolated suppressor mutants that can tolerate toxic serine concentrations by three targeted and non‐targeted genome‐wide screens. All screens as well as genetic complementation in Escherichia coli identified the so far uncharacterized permease YbeC as the major serine transporter of B. subtilis. In addition to YbeC, the threonine transporters BcaP and YbxG make minor contributions to serine uptake. A strain lacking these three transporters was able to tolerate 100 mM serine whereas the wild type strain was already inhibited by 1 mM of the amino acid. The screen for serine‐resistant mutants also identified mutations that result in increased serine degradation and in increased expression of threonine biosynthetic enzymes suggesting that serine toxicity results from interference with threonine biosynthesis.
Bacterial surface polysaccharides are synthesized from lipid-linked precursors at the inner surface of the cytoplasmic membrane before being translocated across the bilayer for envelope assembly. ...Transport of the cell wall precursor lipid II in Escherichia coli requires the broadly conserved and essential multidrug/oligosaccharidyl-lipid/polysaccharide (MOP) exporter superfamily member MurJ. Here, we show that Bacillus subtilis cells lacking all 10 MOP superfamily members are viable with only minor morphological defects, arguing for the existence of an alternate lipid II flippase. To identify this factor, we screened for synthetic lethal partners of MOP family members using transposon sequencing. We discovered that an uncharacterized gene amj (alternate to MurJ; ydaH) and B. subtilis MurJ (murJBs; formerly ytgP) are a synthetic lethal pair. Cells defective for both Amj and MurJBs exhibit cell shape defects and lyse. Furthermore, expression of Amj or MurJBs in E. coli supports lipid II flipping and viability in the absence of E. coli MurJ. Amj is present in a subset of gram-negative and gram-positive bacteria and is the founding member of a novel family of flippases. Finally, we show that Amj is expressed under the control of the cell envelope stress-response transcription factor σ(M) and cells lacking MurJBs increase amj transcription. These findings raise the possibility that antagonists of the canonical MurJ flippase trigger expression of an alternate translocase that can resist inhibition.
A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources ...are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.
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•Construction of two ordered gene deletion mutant libraries of B. subtilis•Defined essential and auxotrophic gene sets in B. subtilis•High-throughput methods for transformation and double-mutant analysis•Genome-wide screening of growth, competence, and sporulation
Koo et al. have constructed two complete ordered single-gene deletion mutant libraries of the model Gram-positive model bacterium, B. subtilis, and developed new high-throughput methodologies to enable facile screening. They have assessed gene essentiality, auxotrophy, competence, and sporulation genome-wide. These libraries and methods provide versatile resources for the study of gene functions, pathway connections, and their regulation.
Essential gene functions underpin the core reactions required for cell viability, but their contributions and relationships are poorly studied in vivo. Using CRISPR interference, we created ...knockdowns of every essential gene in Bacillus subtilis and probed their phenotypes. Our high-confidence essential gene network, established using chemical genomics, showed extensive interconnections among distantly related processes and identified modes of action for uncharacterized antibiotics. Importantly, mild knockdown of essential gene functions significantly reduced stationary-phase survival without affecting maximal growth rate, suggesting that essential protein levels are set to maximize outgrowth from stationary phase. Finally, high-throughput microscopy indicated that cell morphology is relatively insensitive to mild knockdown but profoundly affected by depletion of gene function, revealing intimate connections between cell growth and shape. Our results provide a framework for systematic investigation of essential gene functions in vivo broadly applicable to diverse microorganisms and amenable to comparative analysis.
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•CRISPRi knockdown of essential genes enables discovery of direct antibiotic targets•An essential gene network reveals functional connections between core processes•A cell morphology screen identifies essential genes intimately tied to cell shape•A majority of essential genes show morphological defects as a terminal phenotype
A systematic analysis of all essential genes in Bacillus subtilis using a CRISPR-based knockdown approach established the essential gene network, identified modes of action for antibiotics, and discerned fundamental underpinnings of growth and morphological characteristics of cells.
Gram-negative bacteria can antagonize neighboring microbes using a type VI secretion system (T6SS) to deliver toxins that target different essential cellular features. Despite the conserved nature of ...these targets, T6SS potency can vary across recipient species. To understand the functional basis of intrinsic T6SS susceptibility, we screened for essential Escherichia coli (Eco) genes that affect its survival when antagonized by a cell wall-degrading T6SS toxin from Pseudomonas aeruginosa, Tae1. We revealed genes associated with both the cell wall and a separate layer of the cell envelope, lipopolysaccharide, that modulate Tae1 toxicity in vivo. Disruption of genes in early lipopolysaccharide biosynthesis provided Eco with novel resistance to Tae1, despite significant cell wall degradation. These data suggest that Tae1 toxicity is determined not only by direct substrate damage, but also by indirect cell envelope homeostasis activities. We also found that Tae1-resistant Eco exhibited reduced cell wall synthesis and overall slowed growth, suggesting that reactive cell envelope maintenance pathways could promote, not prevent, self-lysis. Together, our study reveals the complex functional underpinnings of susceptibility to Tae1 and T6SS which regulate the impact of toxin-substrate interactions in vivo.
The vast majority of bacteria, including human pathogens and microbiome species, lack genetic tools needed to systematically associate genes with phenotypes. This is the major impediment to ...understanding the fundamental contributions of genes and gene networks to bacterial physiology and human health. Clustered regularly interspaced short palindromic repeats interference (CRISPRi), a versatile method of blocking gene expression using a catalytically inactive Cas9 protein (dCas9) and programmable single guide RNAs, has emerged as a powerful genetic tool to dissect the functions of essential and non-essential genes in species ranging from bacteria to humans
. However, the difficulty of establishing effective CRISPRi systems across bacteria is a major barrier to its widespread use to dissect bacterial gene function. Here, we establish 'Mobile-CRISPRi', a suite of CRISPRi systems that combines modularity, stable genomic integration and ease of transfer to diverse bacteria by conjugation. Focusing predominantly on human pathogens associated with antibiotic resistance, we demonstrate the efficacy of Mobile-CRISPRi in gammaproteobacteria and Bacillales Firmicutes at the individual gene scale, by examining drug-gene synergies, and at the library scale, by systematically phenotyping conditionally essential genes involved in amino acid biosynthesis. Mobile-CRISPRi enables genetic dissection of non-model bacteria, facilitating analyses of microbiome function, antibiotic resistances and sensitivities, and comprehensive screens for host-microorganism interactions.
Forecasting workloads and responding promptly with resource scaling and migration is critical to optimizing operations and enhancing resource management in cloud environments. However, the diverse ...and dynamic nature of devices within cloud environments complicates workload forecasting. These challenges often lead to service level agreement violations or inefficient resource usage. Hence, this paper proposes an Enhanced Long-Term Cloud Workload Forecasting (E-LCWF) framework designed specifically for efficient resource management in these heterogeneous and dynamic environments. The E-LCWF framework processes individual resource workloads as multivariate time series and enhances model performance through anomaly detection and handling. Additionally, the E-LCWF framework employs an error-based ensemble approach, using transformer-based models and Long-Term Time Series Forecasting (LTSF) linear models, each of which has demonstrated exceptional performance in LTSF. Experimental results obtained using virtual machine data from real-world management information systems and manufacturing execution systems show that the E-LCWF framework outperforms state-of-the-art models in forecasting accuracy.
DNA topoisomerases play essential roles in chromosome organization and replication. Most bacteria possess multiple topoisomerases which have specialized functions in the control of DNA supercoiling ...or in DNA catenation/decatenation during recombination and chromosome segregation. DNA topoisomerase I is required for the relaxation of negatively supercoiled DNA behind the transcribing RNA polymerase. Conflicting results have been reported on the essentiality of the topA gene encoding topoisomerase I in the model bacterium Bacillus subtilis. In this work, we have studied the requirement for topoisomerase I in B. subtilis. All stable topA mutants carried different chromosomal amplifications of the genomic region encompassing the parEC operon encoding topoisomerase IV. Using a fluorescent amplification reporter system we observed that each individual topA mutant had acquired such an amplification. Eventually, the amplifications were replaced by a point mutation in the parEC promoter region which resulted in a fivefold increase of parEC expression. In this strain both type I topoisomerases, encoded by topA and topB, were dispensable. Our results demonstrate that topoisomerase IV at increased expression is necessary and sufficient to take over the function of type 1A topoisomerases.
Cells often mount transcriptional responses and activate specific sets of genes in response to stress-inducing signals such as heat or reactive oxygen species. Transcription factors in the RpoH ...family of bacterial alternative σ factors usually control gene expression during a heat shock response. Interestingly, several α-proteobacteria possess two or more paralogs of RpoH, suggesting some functional distinction. We investigated the target promoters of Rhodobacter sphaeroides RpoH(I) and RpoH(II) using genome-scale data derived from gene expression profiling and the direct interactions of each protein with DNA in vivo. We found that the RpoH(I) and RpoH(II) regulons have both distinct and overlapping gene sets. We predicted DNA sequence elements that dictate promoter recognition specificity by each RpoH paralog. We found that several bases in the highly conserved TTG in the -35 element are important for activity with both RpoH homologs; that the T-9 position, which is over-represented in the RpoH(I) promoter sequence logo, is critical for RpoH(I)-dependent transcription; and that several bases in the predicted -10 element were important for activity with either RpoH(II) or both RpoH homologs. Genes that are transcribed by both RpoH(I) and RpoH(II) are predicted to encode for functions involved in general cell maintenance. The functions specific to the RpoH(I) regulon are associated with a classic heat shock response, while those specific to RpoH(II) are associated with the response to the reactive oxygen species, singlet oxygen. We propose that a gene duplication event followed by changes in promoter recognition by RpoH(I) and RpoH(II) allowed convergence of the transcriptional responses to heat and singlet oxygen stress in R. sphaeroides and possibly other bacteria.
Essential genes are the hubs of cellular networks, but lack of high-throughput methods for titrating gene expression has limited our understanding of the fitness landscapes against which their ...expression levels are optimized. We developed a modified CRISPRi system leveraging the predictable reduction in efficacy of imperfectly matched sgRNAs to generate defined levels of CRISPRi activity and demonstrated its broad applicability. Using libraries of mismatched sgRNAs predicted to span the full range of knockdown levels, we characterized the expression-fitness relationships of most essential genes in Escherichia coli and Bacillus subtilis. We find that these relationships vary widely from linear to bimodal but are similar within pathways. Notably, despite ∼2 billion years of evolutionary separation between E. coli and B. subtilis, most essential homologs have similar expression-fitness relationships with rare but informative differences. Thus, the expression levels of essential genes may reflect homeostatic or evolutionary constraints shared between the two organisms.
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•A universal bacterial system for titrating CRISPRi using partially mismatched sgRNAs•Determined expression-fitness relationships of E. coli and B. subtilis essentialome•Expression-fitness relationships are shared within pathways and between homologs•Shared homeostatic constraints underlie the optimization of essential gene expression
Hawkins and Silvis et al. develop a system for predictably titrating gene expression in bacteria by introducing specific mismatches into CRISPRi sgRNAs. Mismatched sgRNAs enable multiple knockdown levels across many genes in a single experiment. They use this technique to determine the expression-fitness curves of all essential genes in Escherichia coli and Bacillus subtilis, finding that they are shared within pathways and between homologs diverged by ∼2 billion years.
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